1. Technical Field of the Invention
This invention relates generally to oligonucleotide derivatives. More specifically, the present invention relates to an oligonucleotide derivative comprising biotin or 2,4-dinitrobenzene bonded to a moiety other than the base of the nucleotide. The present invention also relates to a method for producing such an oligonucleotide.
2. Description of the Background
Biotin, which is a member of the vitamin B complex and is also called vitamin H, is necessary for the growth of many animals and vegetables. On the other hand, biotin is known to interact potently with avidin in egg white, and this characteristic is put into practical application by utilizing biotin in the form of its derivative such as a biotin-avidin reagent. This is applied as a biochemical reagent for measurement of antigenic density per cell, radioimmunoassay, enzyme immunoassay, etc. Also, a DNA probe having biotin bonded to a nucleic acid has been developed and is now commercially available for diagnosis of infectious and hereditary diseases (Proc. Natl. Acad. Sci. USA, 78, 6633-6637, 1981). The biotin nucleotide derivative in this DNA probe is prepared enzymatically with DNA or RNA as the template by using the biotin derivative of cytidine triphosphate in place of cytidine triphosphate (dCTP).
However, according to our knowledge, a biotin nucleotide derivative thus prepared involves the following problems.
(a) Since biotin is included at the base moiety of the nucleotide, a change occurs in the melting temperature (Tm value) inherent in the oligonucleotide employed.
(b) Synthesis of a cytosine derivative is difficult (in view of the above cited reference).
(c) Synthesis of a DNA having a desired and predetermined base sequence is difficult.
For these reasons, biotin-nucleotide derivatives at the present stage have only a narrow scope of applications and are limited in their usefulness.
Vincent et al have recently developed a DNA probe having 2,4-dinitrophenyl (hereinafter abbreviated as DNP) group bonded to nucleic acid (Nucl. Acids Res., 10, 6787-6796, 1982). They cause a DNP derivative of adenosine triphosphate (ATP) to be incorporated into a DNA strand to be hybridized with a DNA having a complementary base sequence and then detect the objective DNA by adding successively a rabbit antiserum against DNP and peroxide-labelled goat antirabbit I.sub.g G. The DNA strand employed here is a fragment taken from nature.
However, according to our knowledge, the DNP-nucleotide derivative thus prepared involves the following problems.
(a) Since DNP is included at the base moiety of the nucleotide, a change occurs in the melting temperature (Tm value) inherent in the oligonucleotide employed.
(b) Synthesis of a DNA having a desired and predetermined base sequence is difficult.
For these reasons, DNP-nucleotide derivatives at the present stage also have only a narrow scope of applications and are limited in their usefulness.